Gluconeogenesis. Gluconeogenesis / TCA 11/12/2009. Free energy changes in glycolysis 11/13/2009

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Gluconeogenesis Gluconeogenesis / TCA 11/12/2009 Gluconeogenesis is the process whereby precursors such as lactate,

Fasting requires all the glucose to be synthesized from these non-carbohydrate precursors.

Oxaloacetate is the starting material for gluconeogenesis Overview of Glucose Metabolism The metabolic fate of

1 Gluconeogenesis Gluconeogenesis / TCA 11/12/2009 Gluconeogenesis is the process whereby precursors such as lactate, pyruvate, glycerol, and amino acids are converted to glucose. Fasting requires all the glucose to be synthesized from these non-carbohydrate precursors. Most precursors must enter the Krebs cycle at some point to be converted to oxaloacetate. Oxaloacetate is the starting material for gluconeogenesis Overview of Glucose Metabolism The metabolic fate of pyruvate Free energy changes in glycolysis Reaction enzyme ΔG ΔG 1 Hexokinase PGI PFK Aldolase TIM GAPDH+PGK PGM Enolase PK

Gluconeogenesis is not just the reverse of glycolysis Several steps are different so that control of one pathway does not

1 Pyruvate to PEP 2 Fructose 1,6- bisphosphate to Fructose-6- phosphate 3 Glucose-6-Phosphate to Glucose Gluconeogenesis versus

1. Pyruvate carboxylase catalyses the ATP-driven formation of oxaloacetate from pyruvate and CO 2 2.

Pyruvate carboxylase requires biotin as a cofactor Acetyl-CoA regulates pyruvate carboxylase Increases in oxaloacetate

2 Gluconeogenesis is not just the reverse of glycolysis Several steps are different so that control of one pathway does not inactivate the other. However many steps are the same. Three steps are different from glycolysis. 1 Pyruvate to PEP 2 Fructose 1,6- bisphosphate to Fructose-6- phosphate 3 Glucose-6-Phosphate to Glucose Gluconeogenesis versus Glycolysisl Pyruvate is converted to oxaloacetate before being changed to Phosphoenolpyruvate Gluconeogenesis versus Glycolysisl 1. Pyruvate carboxylase catalyses the ATP-driven formation of oxaloacetate from pyruvate and CO 2 2. PEP carboxykinase (PEPCK) concerts oxaloacetate to PEP that uses GTP as a phosphorylating agent. Pyruvate carboxylase requires biotin as a cofactor Acetyl-CoA regulates pyruvate carboxylase Increases in oxaloacetate concentrations increase the activity of the Krebs cycle and acetyl-coa is a allosteric activator of the carboxylase. However when ATP and NADH concentrations are high and the Krebs cycle is inhibited, oxaloacetate goes to glucose. 2

Regulators of gluconeogenic enzyme activity Enzyme Allosteric Allosteric

citrate AMP, F2-6P FBPase AMP, F2-6P PK Alanine F1-6P Inactivates Pyr. Carb.

Glycerol-3-P Activates Glucogon Glycogen is a D-glucose polymer α(1 4)

Breakdown or Glycogenolysis Glycogen Syntheisis Three steps Glycogen

3 Regulators of gluconeogenic enzyme activity Enzyme Allosteric Allosteric Enzyme Protein Inhibitors Activators Phosphorylation Synthesis PFK ATP, citrate AMP, F2-6P FBPase AMP, F2-6P PK Alanine F1-6P Inactivates Pyr. Carb. AcetylCoA PEPCK PFK-2 Citrate AMP, F6P, Pi Inactivates FBPase-2 F6P Glycerol-3-P Activates Glucogon Glycogen is a D-glucose polymer α(1 4) linkages α(1 6) linked branches every 8-14 residues Glycogen Storage Glycogen Breakdown or Glycogenolysis Glycogen Syntheisis Three steps Glycogen phosphorylase Glycogen + Pi <-> glycogen + G1P (n residues) (n-1 residues) Glycogen debranching Phosphoglucomutase 3

Phosphoglucomutase UDP-glucose Pyrophorylase Glycogen Synthase The Citric acid cycle It is called the

It accounts for the majority of carbohydrate, fatty acid and amino acid oxidation.

Amphibolic - acts both catabolically and anabolically 3NAD+ + FAD + GDP + Pi + acetyl-coa 3NADH + FADH

4 Phosphoglucomutase UDP-glucose Pyrophorylase Glycogen Synthase The Citric acid cycle It is called the Krebs cycle or the tricarboxylic and is the hub of the metabolic system. It accounts for the majority of carbohydrate, fatty acid and amino acid oxidation. It also accounts for a majority of the generation of these compounds and others as well. Amphibolic - acts both catabolically and anabolically 3NAD+ + FAD + GDP + Pi + acetyl-coa 3NADH + FADH 2 + GTP + CoA + 2CO 2 The citric acid cycle enzymes are found in the matrix of the mitochondria Substrates have to flow across the outer and inner parts of the mitochondria 4

Nathan Kaplan and Fritz Lipmann discovered Coenzyme A and Ochoa and Lynen showed that acetyl- CoA was

CoA acts as a carrier of acetyl groups Acetyl-CoA is a high energy compound: The ΔG ' for the hydrolysis of its

5 kj mol -1 making it greater than the hydrolysis of ATP Pyruvate dehydrogenase converts pyruvate to acetyl-coa

catalyze two or more sequential steps in a metabolic pathway. Molecular weight of 4,600,000 Da E.

5 Nathan Kaplan and Fritz Lipmann discovered Coenzyme A and Ochoa and Lynen showed that acetyl- CoA was intermediate from pyruvate to citrate. CoA acts as a carrier of acetyl groups Acetyl-CoA is a high energy compound: The ΔG ' for the hydrolysis of its thioester is kj mol -1 making it greater than the hydrolysis of ATP Pyruvate dehydrogenase converts pyruvate to acetyl-coa and CO 2 Pyruvate dehydrogenase A multienzyme complexes are groups of non covalently associated enzymes that catalyze two or more sequential steps in a metabolic pathway. Molecular weight of 4,600,000 Da E. coli yeast Pyruvate dehydrogenase -- E dihydrolipoyl transacetylase --E dihydrolipoyl dehydrogenase--e E2 subunits 24 E1 orange a and b together 12 E3 Red EM based image of the core E2 from yeast pyruvate dh 60 subunits associated as 20 cone-shaped trimers that are verticies of a dodecahedron 5

6 Why such a complex set of enzymes? Covalent modification of eukaryotic pyruvate dehydrogenase 1 Enzymatic reactions rates are limited by diffusion, with shorter distance between subunits a enzyme can almost direct the substrate from one subunit (catalytic ti site) to another. 2. Channeling metabolic intermediates between successive enzymes minimizes side reactions 3. The reactions of a multienzyme complex can be coordinately controlled The five reactions of the pyruvate dehydrogenase multi enzyme complex The enzyme requires five coenzymes and five reactions Pyruvate + CoA + NAD + acetyl-coa + CO 2 + NADH The Coenzymes and prosthetic groups of pyruvate dehydrogenase Domain structure of dihydrolipoyl transacetylase E2 Cofactor Location Function Thiamine Bound to E1 Decarboxylates pyrophosphate pyruvate Lipoic acid Covalently linked Accepts to a Lys on hydroxyethyl E2 (lipoamide) carbanion from TPP CoenzymeA Substrate for E2 Accepts acetyl group from lipoamide FAD (flavin) Bound to E3 reduced by lipoamide NAD + Substrate for E3 reduced by FADH2 6

7 Pyruvate dehydrogenase 1. Pyruvate dh decarboxylates pyruvate using a TPP cofactor forming hydroxyethyl-tpp. 2 The hydroxyethyl group is transferred to the oxidized lipoamide on E2 to form Acetyl dihydrolipoamide-e2 3 E2 catalyzes the transfer of the acetyl groups to CoA yielding acetyl-coa and reduced dihydrolipoamide-e2 4 Dihydrolipoyl dh E3 reoxidizes dihydrolipoamide-e2 and itself becomes reduced as FADH2 is formed 5 Reduced E3 is reoxidized by NAD + to form FAD and NADH The enzymes SH groups are reoxidized by the FAD and the electrons are transferred to NADH Next Lecture Tuesday 11/17/09 Citric Acid Cycle 7

The Citric acid cycle. The Citric Acid Cycle II 11/17/2009. Overview. Pyruvate dehydrogenase

The Citric acid cycle. The Citric Acid Cycle II 11/17/2009. Overview. Pyruvate dehydrogenase The itric acid cycle The itric Acid ycle II 11/17/2009 It is called the Krebs cycle or the tricarboxylic and is the hub of the metabolic system. It accounts for the majority of carbohydrate, fatty acid